Signalling pathways downstream of vascular endothelial growth factor receptors

Vascular endothelial growth factor (VEGF) is an essential angiogenic factor for formation of the embryonic vasculature, and also has important roles in pathological conditions such as diabetic retinopthy, rheumatoid arthritis and cancer. Although the signalling pathways induced by VEGF have been well-studied, the precise molecular mechanisms remain to be determined, particularly with respect to the relative contribution of the individual receptors.;Two main VEGF receptors are expressed in endothelial cells: VEGFR-1 and VEGFR-2. To identify novel effectors downstream of these receptors, their intracellular domains were used as bait to screen yeast two-hybrid cDNA libraries. The association of potential effector proteins with the VEGFRs were assessed in both yeast and mammalian cells, and the identity of the interacting residues was probed using site-directed mutagenesis and peptide competition experiments. These data revealed previously unreported interactions between VEGFRs and WW domain-containing proteins - interactions that could have important implications for the mechanisms regulating VEGF expression.;Analysis of phosphorylation-dependent interactions downstream of VEGFR-1 in mammalian cells, which is generally hindered by the lack of ligand-induced receptor phosphorylation, was enabled by the development of chimeric receptors. In these constructs, the intracellular domains of the VEGFRs were fused downstream of the dimerization domain of the GyrB subunit of bacterial DNA gyrase. The chimeric receptors were significantly activated in response to ligand, thereby enabling studies of this signalling with the putative effectors PKB/Akt, PLC and ERK1.2. Both chimeric receptors were able to activate these effectors. In addition, the observed dependence of some of these effects on a specific tyrosine residue within VEGFR-2 suggested that the chimeric receptors couple to downstream effectors in a manner analogous to that of the full-length receptors